Irwin M. Arias, MD, Head, Unit of Cellular Polarity, Adjunct Scientist1
Victoria Coggin, PhD, Postdoctoral Fellow ²
Barry Elkind, PhD, Postdoctoral Fellow ³
Janet Larkin, PhD, Postdoctoral Fellow ³
Yoshi Wakabayashi, PhD, Postdoctoral Fellow ⁴

In collaboration with Jennifer Lippincott-Schwartz and members of her laboratory, we use live-cell fluorescent, biochemical, genetic, and molecular techniques to study mechanisms responsible for selective trafficking of proteins to the apical domain of hepatocytes and other polarized cells. Our goal is to identify components and regulation of these processes, their role in creating and maintaining cellular polarity, and molecular defects responsible for inheritable and acquired bile- secretory failure (cholestasis).

• Intracellular pathways for trafficking ATP binding cassette transporters to the bile canalicular domain; development of new MDCK cell system for imaging intracellular trafficking
• The role of rab 11a, myosin Vb, and other proteins in canalicular polarity
• Physiologic effect of in vivo expression of adenoviral rab 11a-YFP and myosin Vb-CFP dominant negative constructs in rat liver
• Role of rab 3D in transcytosis
• Regulation of intracellular localization and trafficking of ABC transporters of the G subfamily
• Biology and pathobiology of fenestrae in hepatic endothelial cells
• Function of MRP6 in pseudoxanthoma elasticum
• Role of specific decapeptide in regulating PI 3 kinase activity and in intracellular trafficking of ABC transporters
• Gene expression in cholestasis associated with hyperalimentation
• Molecular pathogenesis of progressive familial intrahepatic cholestasis, type 1
• Effect of hepatitis B replicon expression on BSEP trafficking in WIF-B9 cells
• Sexual dimorphic expression of ABC transporters
• COLLABORATORS

Intracellular pathways for trafficking ATP binding cassette transporters to the bile canalicular domain; development of new MDCK cell system for imaging intracellular trafficking

Wakabayashi, Ortiz5

Previously, we discovered two pathways by which apical membrane proteins traffic from the Golgi to the bile canaliculus in mammalian hepatocytes and polarized WIFB9 cells, which are a hybrid of a rat hepatoma and human fibroblasts. Canalicular ATP binding cassette (ABC) proteins, such as BSEP (bile acid transporter), MRP2 (nonbile acid organic anion transporter), and MDR 1 (organic cation transporter), enter a large intracellular rab 11a-enriched endosomal pool, from which they cycle to the apical plasma membrane. In contrast, single transmembrane proteins, such as cCAM105 and 5´ nucleotidase, traffic from the Golgi to the basolateral plasma membrane domain, from which they undergo transcytosis to the apical membrane. We identified critical roles for tubulin, actin, GGA, hax 2, myosin vb, PI 3-kinase, and rab 11a in the direct trafficking pathway. Live-cell imaging of BSEP-YFP constructs reveals downstream docking sites in the canalicular membrane, sites that we seek to identify.

The role of rab 11a, myosin Vb, and other proteins in canalicular polarity

Wakabayashi, Larkin, Elkind

While studying mechanisms of apical targeting in WIFB9 cells, we observed that rab 11a and myosin Vb are required for canalicular formation. Expression of dominant negative constructs or RNAi prevented polarization and resulted in trafficking patterns found in nonpolarized cells. These observations prompt revision of current polarity concepts and suggest that polarization is initiated upon delivery of rab 11a-, myosin Vb-containing vesicles to the surface, which causes plasma membrane at the site of delivery to differentiate into the apical domain (bile canaliculus).

Physiologic effect of in vivo expression of adenoviral rab 11a-YFP and myosin Vb-CFP dominant negative constructs in rat liver

Veilleux,6 Ortiz,5 Wakabayashi

Using adenoviral YFP and CFP constructs of rab 11a and BSEP, we expanded our in vivo cell-biologic studies in rats. The viral constructs are abundantly expressed in most hepatocytes but not in other cells. Changes in ABC transporter distribution and function were similar to those observed in cell cultures. The in vivo studies provide an exciting opportunity to explore the molecular mechanisms of bile-secretory failure (cholestasis) and the effect of various cholestatic drugs, viruses, diets, and development while suggesting possibilities for the creation of new therapies.

Role of rab 3D in transcytosis

Larkin; in collaboration with Remaley

Transcytosis of membrane proteins in polarized cells is functionally important; however, the underlying molecules and cellular mechanisms and their regulation are poorly understood. Live-cell imaging and biochemical studies suggest that rab 3D may be critical in transcytosis. We are studying this process by using molecular knockdown methodology, expression of dominant negative constructs, and mice in which rab3D has been deleted.

Regulation of intracellular localization and trafficking of ABC transporters of the G subfamily

Elkind; in collaboration with Kruth

Using transfected cell lines, we will expand earlier observations made with MDR-GFP and BSEP-YFP to other ABC transporters, particularly ABCG1 and ABCG2, whose functions and regulation are less well understood. To this end, we produced adenoviral GFP-ABCG2 constructs and dominant negatives and the lentiviral-expressed ABC transporters ABCG1 and ABCG2. We will simultaneously express ABC transporters and RNAi for specific knockdown of gene products involved in trafficking of the ABC transporter in question. To understand the role of the ABCG1 transporter in foam cell production and cholesterol homeostasis, we will also express ABCG1 from a lentiviral vector in human monocyte-derived macrophages and foam cells. ABCG1 is highly induced upon macrophage uptake of LDL cholesterol and causes cholesterol efflux from macrophages directly to HDL, which represents a different mechanism for cholesterol efflux than that used by ABCA1. Our working hypothesis posits that ABCGl regulates cholesterol efflux and that ABCG1 overexpression in response to cholesterol/lipid loading causes macrophage death in the atherosclerotic plaque, resulting in thrombosis.

Biology and pathobiology of fenestrae in hepatic endothelial cells

Coggin; in collaboration with Leapman

Hepatic endothelial cells are heavily fenestrated. Our previous studies indicated that the fenestrae are formed on an actin-myosin-based cytoskeleton and that their contraction can be regulated physiologically. Given that there is no basement membrane in hepatic sinusoids, fenestrae constitute the only barrier between the circulation and the plasma membrane of hepatocytes. Using a newly described hepatic endothelial cell line with regulatable fenestrae, we are exploring the cell biology and physiology of fenestrae. In addition, scanning electron microscopy of liver from mice with deleted caveolin 1 revealed highly abnormal fenestrae with reduced number, size, and configuration. The relation of caveolin 1 to fenestra formation is under study.

Function of MRP6 in pseudoxanthoma elasticum

Arias; in collaboration with Rojkind

MRP6, an ABC transporter restricted to the basalateral plasma membrane of hepatocytes, is mutated in patients with pseudoxanthoma elasticum, a disease caused by impaired elastic tissue in blood vessels, eye, and skin. We are exploring the possibility that the hepatocyte normally secretes an elastase inhibitor into the serum. A sensitive, specific assay for elastase activity serves as a screen for a battery of candidate small peptides that may be biologic substrates for mrp6 and serve as elastase inhibitors in vivo.

Role of specific decapeptide in regulating PI 3 kinase activity and in intracellular trafficking of ABC transporters

Arias; in collaboration with Cantley, Jamney, Leveille-Webster, Kipp

We are exploring the role of a decapeptide that enhances PI 3-kinase activity in cells and in vivo by rendering the substrate phosphatidylinositol 4,5-bisphosphate (PI45P2) more susceptible to the enzyme. Our studies reveal that 3′ phosphoinositides are required for trafficking of ABC transporters and for their activity in the plasma membrane. Furthermore, we showed that the rhodamine-conjugated decapeptide is a potent choleretic agent in vivo; thus, it may be useful therapeutically.

Gene expression in cholestasis associated with hyperalimentation

Arias; in collaboration with Friedman, Gottesman

We are exploring gene expression patterns in clinical and experimental cholestasis associated with hyperalimentation. The Gottesman laboratory has developed an array procedure that permits examination of all known hepatocyte transporters, drug metabolizing enzymes, and other critical genes.

Molecular pathogenesis of progressive familial intrahepatic cholestasis, type 1

Arias; in collaboration with Harris, Schneider

We are studying the cellular and molecular pathogenesis of progressive familial intrahepatic cholestasis, type 1 (PFIC 1). FIC1 encodes a P-type ATPase, which, as we previously showed, functions as an aminophospholipid flippase in the basalateral plasma membrane of hepatocytes and small intestinal cells. FIC1 regulates FXR, a nuclear transcription factor, which, in turn, regulates the activity of BSEP and other apical ABC transporters. Using molecular and imaging techniques, we seek to elucidate how the P-ATPase and its lipid traffic regulate bile acid secretion.

Effect of hepatitis B replicon expression on BSEP trafficking in WIF-B9 cells

Arias; in collaboration with Rice

Infection of hepatocytes with hepatitis B or C viruses frequently produces prolonged cholestasis without cellular necrosis. We propose that viral replication is associated with altered intracellular trafficking of canalicular ABC transporters, particularly Bsep. In collaboration with Charles Rice and colleagues, we are investigating the effect of several HBV replicons on trafficking of ABC transporters in WIF-B9 cells.

Sexual dimorphic expression of ABC transporters

Arias; in collaboration with Simon

We are examining the mechanism responsible for sexual dimorphic expression of mrp2 and other ABC transporters in mice. Our studies reveal that the process is mediated by growth hormone and modified by thyroid hormone.

1 Assistant to the Director, Intramural Program, NIHGRI, Bethesda, MD; Professor of Physiology and Medicine, Tufts School of Medicine, Boston, MA
2 On sabbatical leave from ANZAC Research Institute, University of Sydney, Australia, until June 2006
3 Left laboratory during 2006.
4 Tufts School of Medicine, Boston, MA
5 Daniel Ortiz, PhD, Research Assistant Professor, Tufts School of Medicine, Boston, MA
6 Michael Veilleux, MA, Research Technician, Tufts School of Medicine, Boston, MA

COLLABORATORS

Suresh Ambudkar, PhD, Laboratory of Cell Biology, NCI, Bethesda, MD
Lewis Cantley, PhD, Harvard Medical School, Cambridge, MA
Thomas Friedman, MD, Georgetown Medical School, Washington, DC
Michael Gottesman, MD, Laboratory of Cell Biology, NCI, Bethesda, MD
Matt Harris, PhD, Avastra, Inc., Sydney, Australia
Paul Jamney, PhD, University of Pennsylvania School of Medicine, Philadelphia, PA
Helmut Kipp, PhD, Universität Würzburg, Würzburg, Germany
Howard Kruth, PhD, Cardiovascular Branch, NHLBI, Bethesda, MD
Richard Leapman, PhD, Division of Bioengineering Physical Science, NIH, Bethesda, MD
Cynthia Leveille-Webster, DVM, Tufts School of Veterinary Medicine, Boston, MA
Alan Remaley, PhD, Department of Laboratory Medicine, Warren G. Magnuson Clinical Center, NIH, Bethesda, MD
Charles Rice, PhD, The Rockefeller University, New York, NY
Marcos Rojkind, MD, PhD, George Washington University, Washington, DC
Benjamin Schneider, MD, Mount Sinai Medical School, New York, NY
Franz Simon, MD, University of Colorado School of Medicine, Denver, CO

For further information, contact ariasi@mail.nih.gov.